The riddle of organogenesis or how the organs and tissues of the body are formed has been ranked as one of the most important unanswered questions in developmental biology. Although organogenesis is usually studied during embryogenesis, the regeneration of tissues and organs in adult organisms provide alternate organogenetic models. The sea cucumber Holothuria glaberrima, provides a model system where the formation of the intestine can be studied during regeneration. These coelomate organisms, members of the phylum Echinodermata, are closely related to the chordates and exhibit an impressive ability to regenerate complex structures. The proposed research focuses on the molecular aspects of regeneration, specifically on the genes that are important for the regeneration process to occur. The long term goal of our laboratory is to characterize the genes involved and their temporal and spatial pattern of expression. Although we have already identified and characterized several genes involved in holothurian intestinal regeneration, the present proposal will increase exponentially this genetic characterization by using modern genomic techniques. Initially, genes from a H. glaberrima regenerating intestine library will be identified and partially sequenced. Their homology to genes in the database will be determined and unique genes will be selected. Microarrays will be prepared with these unique sequences and probed for regeneration-induced genes. Genes that are preferentially expressed during regeneration will be determined by comparing the expression pattern with probes obtained from non-regenerating animals and from late-stage regeneration. Regeneration specificity of gene sequences will be validated with RT-PCR and in situ hybridization. A selected group of genes will be submitted to further analyses, including a detailed analysis of the gene temporal and spatial expression and the identification of their putative role. Functional studies will be done by interfering with the expression of the gene, or of the gene product, depending on the identified molecule. The outcome of these experiments is of great importance in understanding the formation of organs in deuterostomes. Moreover, the genes identified and characterized in our project will provide key information to explore regenerative processes in adult vertebrates, including humans.